Generation of two induced pluripotent stem cell lines (LVPEIi007-B, LVPEIi008-B) from patients harboring homozygous mutation in ABCA4 (c.6088C>T) using non-integrative Sendai virus-based approach.

Mutations in ABCA4 gene leads to the most common form of an inherited retinal disease namely, the Stargardt disease, type 1. Here, we report the generation of two different patient-specific induced pluripotent stem cell lines (LVPEIi007-B and LVPEIi008-B), carrying an identical homozygous mutation, (c.6088C>T) within the exon 44 of ABCA4 gene. These lines were generated by the reprogramming of patient-specific dermal fibroblasts, using the integration-free, Sendai viral vectors. Both lines were stably expanded and expressed the stemness and pluripotency markers, differentiated into cell types of all three germ layers, and maintained a normal karyotype.

Generation of two induced pluripotent stem cell lines (LVPEIi004-A and LVPEIi005-A) from probands with Leber Congenital Amaurosis 2 (LCA2) and harboring mutations in RPE65.

Leber Congenital Amaurosis 2 is an early onset retinal dystrophy that occurs due to mutation in RPE65 gene. Here, we report the generation of two patient specific induced pluripotent stem cell lines harboring nonsense mutations in exon 7 (c.646A > T) and exon 9 (c.992G > A) of RPE65 gene, respectively, which leads to premature translational termination and formation of defective protein. These lines were generated by the reprogramming of human dermal fibroblast cells using integration-free, episomal constructs expressing stemness genes. The stable lines maintained a normal karyotype, expressed the key stemness factors, underwent trilineage differentiation, and maintained their genetic identity and genomic integrity.

Generation and characterization of three CRISPR/Cas9 edited RB1 null hiPSC lines for retinoblastoma disease modelling.

Complete loss of RB1 causes retinoblastoma. Here, we report the generation of three RB1-/- iPSC lines using CRISPR/Cas9 based editing at exon 18 of RB1 in a healthy control hiPSC line. The edited cells were clonally expanded, genotyped and characterized to establish the mutant lines. Two of the mutant lines are compound heterozygous, with different in-del mutations in each of their alleles, while the third mutant line is homozygous, with identical edits in both alleles. All lines maintained their stemness, pluripotency, formed embryoid bodies with cell types of all three lineages, displayed a normal karyotype and lost RB1 expression.

Generation of Leber congenital amaurosis, type 12 patient-specific induced pluripotent stem cell line (LVPEIi006-A), harboring a homozygous mutation in RD3.

Leber congenital amaurosis (LCA) is a congenital, early onset, autosomal recessive inherited retinal disease (IRD). This report describes an LCA12 patient-specific iPSC line (LVPEIi006-A), generated by the reprogramming of dermal fibroblasts using integration-free episomal plasmids.This disease-specific iPSC model carries a homozygous point mutation in RD3, within the donor splice site at the end of exon 2 (c.296 + 1G > A). The stable line at passage 15 has displayed a normal colony morphology, expressed multiple stemness and pluripotency markers, lost all transgenes, differentiated into cell types of all three germ layers, and maintained a normal karyotype.

Generation of an induced pluripotent stem cell line (LVPEIi002-A) with heterozygous RB1 mutation using peri-orbital fat derived mesenchymal cells of a patient with inherited retinoblastoma.

Retinoblastoma is a pediatric intraocular cancer caused by biallelic inactivation of RB1 gene in retinal progenitor cells. Here, we report the generation of a patient-specific induced pluripotent stem cell (iPSC) line (LVPEIi002-A) from a patient diagnosed with retinoblastoma and showing familial inheritance of a nonsense mutation (c.1735C > T) within exon 18 of one of the two alleles. This RB1+/- iPSC line, LVPEIi002-A was generated by reprogramming the peri-orbital fat tissue derived mesenchymal cells and was stably expanded and characterized. It maintains the stemness, pluripotency, normal karyotype, and forms embryoid bodies comprising of all three lineage committed progenitor cells.

Taking the leap toward human-specific nonanimal methodologies: The need for harmonizing global policies for microphysiological systems.

With a recent amendment, India joined other countries that have removed the legislative barrier toward the use of human-relevant methods in drug development. Here, global stakeholders weigh in on the urgent need to globally harmonize the guidelines toward the standardization of microphysiological systems. We discuss a possible framework for establishing scientific confidence and regulatory approval of these methods.

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

Delivery strategies for CRISPR/Cas genome editing tool for retinal dystrophies: challenges and opportunities.

CRISPR/Cas, an adaptive immune system in bacteria, has been adopted as an efficient and precise tool for site-specific gene editing with potential therapeutic opportunities. It has been explored for a variety of applications, including gene modulation, epigenome editing, diagnosis, mRNA editing, etc. It has found applications in retinal dystrophic conditions including progressive cone and cone-rod dystrophies, congenital stationary night blindness, X-linked juvenile retinoschisis, retinitis pigmentosa, age-related macular degeneration, leber's congenital amaurosis, etc. Most of the therapies for retinal dystrophic conditions work by regressing symptoms instead of reversing the gene mutations. CRISPR/Cas9 through indel could impart beneficial effects in the reversal of gene mutations in dystrophic conditions. Recent research has also consolidated on the approaches of using CRISPR systems for retinal dystrophies but their delivery to the posterior part of the eye is a major concern due to high molecular weight, negative charge, and in vivo stability of CRISPR components. Recently, non-viral vectors have gained interest due to their potential in tissue-specific nucleic acid (miRNA/siRNA/CRISPR) delivery. This review highlights the opportunities of retinal dystrophies management using CRISPR/Cas nanomedicine.

Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.

Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have hindered disease treatments. Here, we show that ABCA4 knockout and induced pluripotent stem cell-derived RPE (STGD1-iRPE) from patients with Stargardt differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity-indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are lowered by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.

Gene therapy in retinal diseases: A review.

Over 2 million people worldwide are suffering from gene-related retinal diseases, inherited or acquired, and over 270 genes have been identified which are found to be responsible for these conditions. This review article touches upon the mechanisms of gene therapy, various enzymes of the visual cycle responsible for different genetic diseases, Luxturna-the first US Food and Drug Administration (FDA)-approved therapeutic gene product, and several ongoing trials of gene therapy for age-related macular degeneration. Gene therapy has tremendous potential for retinal conditions due to its ease of accessibility, immune-privileged status, and tight blood-retinal barriers, limiting systemic side effects of the drug. In recent years, advances in gene therapy in retinal conditions have increasing significantly, with progress in cell-specific targeting and transduction efficiency of gene products through the use of adeno-associated viral vectors (AAVs), suggesting that even greater success in future clinical trials is possible.

Expression of a novel brain specific isoform of C3G is regulated during development.

Mice lacking C3G (RapGEF1), a ubiquitously expressed protein essential for neuronal differentiation, show multiple defects in brain development. Function of C3G in neurogenesis is poorly defined. Here, we identify brain specific expression of a novel C3G isoform in mice and humans. This isoform has an insert in the Crk-binding region, generating a polypeptide of 175 kDa, unlike the previously known 140 kDa form expressed in all other tissues. In the adult mouse brain, C3G expression is seen in neurons, but was not detectable in GFAP-positive cells. C3G levels were high in the CA3 region of hippocampus and in mitral cells of olfactory bulb. Neural progenitor cells positive for Doublecortin and Nestin, show expression of C3G. During development, C3G is expressed in precursor cells prior to their differentiation into mature neurons or astrocytes. The 175 kDa as well as 140 kDa forms are seen in embryonic mouse brain, while only the 175 kDa variant is seen in post-natal brain. Human cerebral organoids generated from induced pluripotent stem cells predominantly expressed the 140 kDa polypeptides, and the 175 kDa isoform appeared upon maturation. This study describes developmental regulation and neuronal expression of a brain specific isoform of C3G, a molecule essential for normal development of the mammalian brain.

Learning curve of a trained vitreo-retinal surgeon in sub-retinal injections in a rat model: Implications for future clinical trials.

Purpose: The sub-retinal injections are not very commonly performed procedures in vitreoretina, but form a crucial step in any cell replacement therapy for retinal diseases. The purpose of this study is to describe the learning curve of a trained vitreo-retinal surgeon in sub-retinal injections in a rat model and its implications in future clinical trials. Methods: This is an in-vivo retrospective animal study using Wistar rats. All ARVO guidelines regarding animal handling were followed. After anesthetization, aspectic preparation and dilating the pupils with 1% tropicamide eye drops, subretinal injection of 10 µl saline was done via a limbal entry. Data recorded included time taken for the procedure, success of injection, associated complications, post-operative infections and complications. The rats were followed up for 1 month post procedure. A trend analysis was done for the above factors to look for improvement in ease of procedure, reduction in procedure time and reduction in complications for the clinician using a novel objective scale. Results: About 20 eyes were studied. Mean weight of the rats was 188 ± 12.82 gram. Mean time taken for the procedure was 14.1 ± 5.07 minutes. There was a significant inverse co-relation between the serial number of the eye and time taken for the procedure (r = -0.89, P < 0.0001). Comparative complications noted between the first ten and the last ten eyes were: conjunctival tear 30% versus 10% (P = 0.27), lens touch 50% versus 10% (P = 0.05), subretinal hemorrhage 40% versus 0% (P = 0.13), vitreous loss 30% versus 0% (P = 0.06). The successful subretinal injection without intraocular complications was achieved in 40% versus 90% (P = 0.02). There was a significant co-relation between the serial number of the eye and ease of the procedure (r = 0.87, P < 0.0001). Post operatively none of the eyes had any infection. Six eyes (12%) developed cataract and 3 eyes (6%) had non-resolving retinal detachment at the last examination visit. Conclusion: Subretinal injections in rats have a definite learning curve even for a trained vitreoretinal surgeon. This should be accounted for and resources allocated accordingly to achieve good technical comfort and negate confounding by the surgeon factor in the results of future clinical trials.

Therapeutic avenues for hereditary forms of retinal blindness.

Hereditary retinal diseases, known as retinal degenerations or dystrophies, are a large group of inherited eye disorders resulting in irreversible visual loss and blindness. They develop due to mutations in one or more genes that lead to the death of the retinal photoreceptor cells. Till date, mutations in over 200 genes are known to be associated with all different forms of retinal disorders. The enormous genetic heterogeneity of this group of diseases has posedmany challenges in understanding the mechanisms of disease and in developing suitable therapies. Therapeutic avenues that are being investigated for these disorders include gene therapy to replace the defective gene, treatment with neurotrophic factors to stimulate the growth of photoreceptors, cell replacement therapy, and prosthetic devices that can capture light and transmit electrical signals through retinal neurons to the brain. Several of these are in process of human trials in patients, and have shown safety and efficacy of the treatment. A combination of approaches that involve both gene replacement and cell replacement may be required for optimum benefit.

Generating minicorneal organoids from human induced pluripotent stem cells.

Corneal epithelial stem cells residing within the annular limbal crypts regulate adult tissue homeostasis. Autologous limbal grafts and tissue-engineered corneal epithelial cell sheets have been widely used in the treatment of various ocular surface defects. In the case of bilateral limbal defects, pluripotent stem cell (PSC)-derived corneal epithelial cells are now being explored as an alternative to allogeneic limbal grafts. Here, we report an efficient method to generate complex three-dimensional corneal organoids from human PSCs. The eye field primordial clusters that emerged from differentiating PSCs developed into whole eyeball-like, self-organized, three-dimensional, miniature structures consisting of retinal primordia, corneal primordia, a primitive eyelid-like outer covering and ciliary margin zone-like adnexal tissues in a stepwise maturation process within 15 weeks. These minicorneal organoids recapitulate the early developmental events in vitro and display similar anatomical features and marker expression profiles to adult corneal tissues. They offer an alternative tissue source for regenerating different layers of the cornea and eliminate the need for complicated cell enrichment procedures.

Surgical Management of Bilateral Limbal Stem Cell Deficiency.

Limbal stem cell deficiency (LSCD) is now established as a distinct entity with a spectrum of clinical manifestations. Bilateral LSCD presents a unique set of challenges to the clinician dealing with ocular surface disease, due to the underlying causes, clinical presentation, and adnexal status, as well as lack of a source of autologous limbal stem cells. Various surgical modalities have been described to achieve visual rehabilitation in patients with bilateral LSCD. These can primarily be divided into cell-based therapies and implantation of keratoprostheses. In this review, the surgical options for management of bilateral LSCD, including autologous and allogeneic cell-based therapies and different types of keratoprostheses are described and classified. The indications, prerequisites, technique, results and complications of each modality are discussed. Based on the status of the ocular surface, an algorithm for choosing appropriate surgical management for vision restoration in bilateral LSCD has been proposed.

Enriched Cultures of Retinal Cells From BJNhem20 Human Embryonic Stem Cell Line of Indian Origin.

PURPOSE: To test the retinal differentiation potential and to establish an optimized protocol for enriching retinal cells from an Indian origin, human embryonic stem cell (hESC) line, BJNhem20. METHODS: The BJNhem20 cells were cultured and expanded under feeder-free culture conditions. Differentiation was initiated by embryoid body (EB) formation and were cultured on Matrigel in neural induction medium (NIM) for 1 week and further maintained in retinal differentiation medium (RDM). After 1 month, the neuro-retinal progenitor clusters located at the center of pigmented retinal patches were picked and cultured as suspended neurospheres in RDM for 3 days and subsequently on Matrigel in neuro-retinal medium. The mildly pigmented, immature retinal pigmented epithelial (RPE) cells were picked separately and cultured on Matrigel in RPE medium (RPEM). After 1 week, the confluent neuro-retinal and RPE cultures were maintained in RDM for 2 to 3 months and characterized by immunofluorescence and RT-PCR. RESULTS: The BJNhem20 cells efficiently differentiated into both neuro-retinal and RPE cells. The early retinal progenitors expressed Nestin, GFAP, Pax6, Rx, MitfA, Chx10, and Otx2. Neuro-retinal cells expressed the neural markers, Map2, ß-III tubulin, acetylated tubulin and photoreceptor-specific markers, Crx, rhodopsin, recoverin, calbindin, PKC, NeuroD1, RLBP1, rhodopsin kinase, PDE6A, and PDE6C. Mature RPE cells developed intense pigmentation within 3 months and showed ZO-1 and Phalloidin staining at cell-cell junctions and expressed RPE65, tyrosinase, bestrophin1, Mertk, and displayed phagocytic activity. CONCLUSIONS: This study confirms the retinal differentiation potential of BJNhem20 cells and describes an optimized protocol to generate enriched populations of neuro-retinal and RPE cells.

Spatial distribution of niche and stem cells in ex vivo human limbal cultures.

Stem cells at the limbus mediate corneal epithelial regeneration and regulate normal tissue homeostasis. Ex vivo cultured limbal epithelial transplantations are being widely practiced in the treatment of limbal stem cell deficiency. In this report, we examined whether the limbal niche cells that nurture and regulate epithelial stem cells coexist in ex vivo limbal cultures. We also compared the inherent differences between explant and suspension culture systems in terms of spatial distribution of niche cells and their effect on epithelial stem cell proliferation, migration, and differentiation in vitro. We report that the stem cell content of both culture systems was similar, explaining the comparable clinical outcomes reported using these two methods. We also showed that the niche cells get expanded in culture and the nestin-positive cells migrate at the leading edges to direct epithelial cell migration in suspension cultures, whereas they are limited to the intact niche in explant cultures. We provide evidence that C/EBPδ-positive, p15-positive, and quiescent, label-retaining, early activated stem cells migrate at the leading edges to regulate epithelial cell proliferation in explant cultures, and this position effect is lost in early suspension cultures. However, in confluent suspension cultures, the stem cells and niche cells interact with each another, migrate in spiraling patterns, and self-organize to form three-dimensional niche-like compartments resembling the limbal crypts and thereby reestablish the position effect. These 3D-sphere clusters are enriched with nestin-, vimentin-, S100-, and p27-positive niche cells and p15-, p21-, p63α-, C/EBPδ-, ABCG2-, and Pax6-positive quiescent epithelial stem cells.

Transforming ocular surface stem cell research into successful clinical practice.

It has only been a quarter of a century since the discovery of adult stem cells at the human corneo-scleral limbus. These limbal stem cells are responsible for generating a constant and unending supply of corneal epithelial cells throughout life, thus maintaining a stable and uniformly refractive corneal surface. Establishing this hitherto unknown association between ocular surface disease and limbal dysfunction helped usher in therapeutic approaches that successfully addressed blinding conditions such as ocular burns, which were previously considered incurable. Subsequent advances in ocular surface biology through basic science research have translated into innovations that have made the surgical technique of limbal stem cell transplantation simpler and more predictable. This review recapitulates the basic biology of the limbus and the rationale and principles of limbal stem cell transplantation in ocular surface disease. An evidence-based algorithm is presented, which is tailored to clinical considerations such as laterality of affliction, severity of limbal damage and concurrent need for other procedures. Additionally, novel findings in the form of factors influencing the survival and function of limbal stem cells after transplantation and the possibility of substituting limbal cells with epithelial stem cells of other lineages is also discussed. Finally this review focuses on the future directions in which both basic science and clinical research in this field is headed.

Unilateral partial limbal stem cell deficiency: contralateral versus ipsilateral autologous cultivated limbal epithelial transplantation.

PURPOSE: To report the outcomes of autologous cultivated limbal epithelial transplantation using the healthy part of the affected eye or the fellow eye as a source of limbal stem cells in patients with unilateral, partial limbal stem cell deficiency (LSCD). DESIGN: Retrospective, nonrandomized, interventional case series. METHODS: setting: L. V. Prasad Eye Institute, Hyderabad, India. study population: Patients with unilateral, partial LSCD who underwent autologous cultivated limbal epithelial transplantation between 2001 and 2011. intervention: The limbal biopsy was taken either from the healthy part of the limbus of the same eye (ipsilateral group) or from the healthy fellow eye (contralateral group). Cells were cultivated using a xeno-free explant culture technique, and cultivated cells were transplanted onto the affected surface. primary outcome measure: Success of cultivated limbal epithelial transplantation, defined as a completely epithelialized, avascular, and clinically stable corneal surface. RESULTS: Seventy eyes of 70 patients were studied. The mean follow up was 17.5 ± 7 months. In 34 eyes the limbal biopsy was taken from the ipsilateral eye and in the remaining 36 eyes from the contralateral eye. Clinical success was achieved in 70.59% of eyes in the ipsilateral group and 75% of eyes in the contralateral group (P = .79). Limbal transplant survival rates at the final follow-up visit were 65.1% ± 0.09% in the ipsilateral group and 53.6% ± 0.12% in the contralateral group (P = .74). CONCLUSIONS: Ocular surface restoration in partial LSCD is possible with cell-based therapy. Outcomes are similar irrespective of whether the limbal biopsy is taken from the healthy part of the ipsilateral eye or the contralateral eye.